Mesenchymal stem/progenitor cells (MSCs) hold considerable potential for a wide range of tissue regeneration therapies. While their differentiation capacity has been demonstrated extensively, mechanisms that control their plasticity remain poorly understood. Observations by our laboratories and others have shown that proliferating MSCs express lineage-associated molecules prior to induction of differentiation. This has led to a suggested model of differentiation where commitment to a specific cell type results from the combined effects of increased expression along the induced pathway and repression of genes related to other lineages. MicroRNAs are endogenously expressed, small RNAs that may regulate this process. By acting as transcriptional repressors, they have recently emerged as regulators of cellular differentiation in cancer and stem cells. In this proposal we will investigate the role of specific microRNAs and their gene targets on MSC differentiation. We have performed microRNA profiling on MSCs undergoing osteogenic and chondrogenic differentiation and identified miR-130b, miR-432 and miR-559 as differentially expressed miRNAs among both lineages. Functional studies in MSCs suggest multilineage regulation by miR-130b; its overexpression enhanced the osteoblast phenotype and repressed smooth muscle differentiation. Differential gene expression analysis in miR-130b transfected MSCs has identified several mRNA targets that could serve as control points for osteo- and myogenic differentiation. Building on these findings, Aim 1 of this proposal will identify direct targets of miR-130b and assess their role in osteo-, chondro- and myogenic differentiation. The same approach will be followed to assess the functional effects and gene targets of miR-432 and -559. In Specific Aim 2, we will investigate whether the functional effects of these microRNAs are replicated in vivo. MicroRNA overexpression and inhibition experiments will be performed by systemic delivery of lentiviral vectors encoding mimics and hairpin inhibitors to developing mouse embryos. At specific developmental stages embryonic tissues will be evaluated for bone, cartilage and smooth muscle formation. Successful completion of this study will 1) determine how miRNA-mediated gene silencing in mesenchymal cells alters commitment to osteo- chondro- and myogenic lineages and 2) identify potential regulatory roles for these specific microRNAs during musculoskeletal development. This should improve our current understanding of mesenchymal stem cell differentiation and aid future cell-based therapies for musculoskeletal repair. PUBLIC HEALTH RELEVANCE: Bone and cartilage repair, following injury or disease, may be facilitated by the transplantation of stem/progenitor cells. Successful repair requires the generation of stable bone/cartilage cells from implanted stem cells through the process of differentiation. This project will study the roles of small regulatory RNAs (microRNAs) in controlling the differentiation pathways of stem cells towards specific cell types. This should uncover novel mechanisms that can be exploited for therapeutic use in cartilage defect repair and fracture healing.